HSP90 multi-functionality in cancer

The 90-kDa heat shock proteins (HSP90s) are molecular chaperones essential for folding, unfolding, degradation and activity of a wide range of client proteins. HSP90s and their cognate co-chaperones are subject to various post-translational modifications, functional consequences of which are not fully understood in cancer. Intracellular and extracellular HSP90 family members (HSP90α, HSP90β, GRP94 and TRAP1) promote cancer by sustaining various hallmarks of cancer, including cell death resistance, replicative immortality, tumor immunity, angiogenesis, invasion and metastasis. Given the importance of HSP90 in tumor progression, various inhibitors and HSP90-based vaccines were developed for the treatment of cancer. Further understanding of HSP90 functions in cancer may provide new opportunities and novel therapeutic strategies for the treatment of cancer.


Introduction
Heat shock protein 90 (HSP90) chaperone machinery plays a critical role in protein folding, unfolding, degradation and maturation processes (1,2).HSP90 chaperones interact with a large and diverse group of client proteins, many of which are important regulators of tumorigenesis, immune suppression, invasion and metastasis (3).HSP90s are primarily located in cytosol, endoplasmic reticulum, and mitochondria (4), but also have been found in the extracellular space associated with tumor progression and unfavorable clinical outcome (5).Overexpression of HSP90s has been implicated in survival and proliferation of tumor cells (6), which was further supported by the finding that HSP90s are upregulated in response to apoptotic stimuli, such as UV, sodium arsenite and doxorubicin (6)(7)(8).In addition, Kruta et al. demonstrated that ex vivo culture stress and aging also induce heat shock response by activating heat shock factor -1 (HSF-1) (9)(10)(11).
In this Review, we focus on the role of HSP90 chaperone machinery in sustaining various hallmarks of cancer and exploring the potential of HSP90 as anti-cancer therapeutic targets.

The HSP90 structure and conformational cycle
Each HSP90 monomer consists of amino-terminal domain (NTD) that is connected to a middle domain (MD) by a linker, and a C-terminal domain (CTD) (Figure 1) (13).In the absence of ATP, HSP90 mainly adopts an open V-shaped conformation (13).ATP binding leads to the conformational change in NTD involving closure of the lids, which is followed by the NTD dimerization and twisting of HSP90 monomers for the efficient ATP hydrolysis (closed conformation) (Figure 1) (13-16).Various co-chaperones assist HSP90 throughout conformational cycle (13).HSP70/HSP90organizing protein (HOP), also known as stress-inducible phosphoprotein 1 (STIP1) and cell division cycle 37 homologue (CDC37) inhibit HSP90 structural changes, whereas activator of HSP90 ATPase homologue 1 (Aha1) accelerates the formation of closed ATP-bound conformation (13,16).Prostaglandin E synthase 3 (PTGES3/p23) acts as a co-chaperone slowing the ATPase cycle by stabilizing the closed conformation that is committed to ATP hydrolysis (13, 17) (Figure 1).

HSP90 secretion into the extracellular milieu
Elevated HSP90 level was detected in plasma/serum in patients with cancer, including liver cancer (45), advanced staged colorectal cancer (46,47), lung cancer (48), acute myeloid leukemia (49), hepatocellular carcinoma (50).Extracellular HSP90s may affect other cells by modulating intercellular signaling when released via EVs (51).EVs play important roles in intercellular communication, regulating a range of biological processes.Given the ability of EVs to carry and transfer tumorigenic factors between cells, EVs have been explored as therapeutic targets, novel drug delivery vehicles, biomarkers and standalone therapeutics in cancer research (52).HSP90s and their co-chaperones have been found in EVs isolated from patients with melanoma (53-55), glioblastoma (56), pancreatic cancer (57), prostate cancer (58), bladder cancer (59), lung cancer (60) and papillary thyroid cancer (61) [reviewed in (62)].Lauwers and colleagues demonstrated that HSP90 in Drosophila regulates the membrane deformation and exosome release (63).Subsequent study demonstrated that HSP90a is located on the surface of exosomes and the monoclonal antibody against HSP90a inhibits the pro-motility activity of tumor-secreted exosomes (64).

HSP90 functions in the hallmarks of cancer
Being abundantly expressed in cancer, HSP90s promote growth and survival of tumor cells by regulating a wide range of processes.Here, we will explore HSP90 involvement in the hallmarks of cancer a model of multi-step cancer development established by Hanahan and Weinberg (65, 66) (Figure 2).

HSP90 and tumor immunity
In 1986 Ullrich and colleagues identified HSP90 as a highly abundant cytosolic and surface tumor-transplantation antigen in methylcholanthrene-induced tumors (Meth A) (68).At the same time Srivastava et al. isolated tumor rejection antigens from the membrane and cytosol fractions of Meth A and CMS5 which was later recognized as ER HSP90 homolog, glucose-regulated protein 94 (GRP94/HSP90B1/gp96/ERp99/Endoplasmin) (69, 70).HSP90s isolated from tumors have been shown to elicit potent anti-tumor  (2,74).This is also supported by the finding that downregulation of heat shock factor (HSF-1) or HSP90 associates with a defective cross-presentation by DCs (75).Furthermore, it has been shown that HSP90 inhibitor reduces the translocation of antigens into the cytosol whereas HSP90AA1 knockdown leads to a loss of proteolytic intermediates and reduced presentation of peptide-MHC I complexes on the cell surface (76,77).Subsequent studies demonstrated that low-level inhibition of HSP90 diversifies the peptide MHC class I repertoire on tumor cells (78).HSP90 inhibitor also showed to decrease MHC II antigen presentation by IFNg-treated APCs (79).Altogether, these data show that HSP90 is critical for MHC I and MHC II class antigen presentation.Apart from antigen presentation, HSP90 is also critical for the phenotype and functional activity of immune cells.In this regard, Bae and colleagues demonstrated that HSP90 inhibitor downregulates CD3, CD8, CD25, CD28, CD40L and ab on the surface of T cells and activating receptors, including CD2, CD11a, CD94, NKp30, NKp44, NKp46, KARp50.3 on NK cells (80).We and others show that HSP90 deficiency impairs NK and T cell proliferation, cytotoxicity and IFNg production (80)(81)(82)(83).By contrast, HSP90 ER homolog GRP94 stimulates NK cells indirectly via APCs (84).On DCs, GRP94 acts via Toll-like receptor 2 (TLR-2) and TLR-4 inducing the expression of CD86 and IL-12 and TNF-a production (85,86).In T regs, GRP94 upregulates Foxp3, IL-10 and TGF-b1 via TLR-2/4-mediated NF-kB activation (87).Interaction of GRP94 with TLR is critical for the activation of cytotoxic T cells response (88).Additionally, GRP94 also induces NLRP3 inflammasome activation and IL-1b production in murine APCs via K + efflux (89).HSP90a on the tumor-cell released autophagosomes (TRAPs) stimulate IL-6 release by CD4+ T cells via TLR2-MyD88-NF-kB pathway (90).Autocrine IL-6 further promotes the production of IL-10 and IL-21 by CD4 + T cells via STAT3, enhancing metastasis (90).It has also been shown that the production of HSP90a, IL-8 and IL-6 by macrophages induces JAK2-STAT3 pathway, supporting invasion and migration in pancreatic ductal epithelial cells (91).On the other hand, cytokines may also induce HSP90 expression, which further enhance their pro and anti-inflammatory activities (Figure 3) (92).Unlike HSP90AA1, HSP90AB1 and HSP90B1, TRAP1 could only be induced by IL-18 in NK cells and IL-3 in conventional DC2 (cDC2) cells (92).Collectively, these studies show that there is an important interplay between HSP90 and cytokines, which should be further explored in the context of cancer.HSP90 family members also play important roles in the regulation of immune checkpoints.Zavareh and colleagues demonstrated that HSP90 inhibitors downregulate PD-L1 mRNA level and surface expression by suppressing HSP90 clients c-Myc and signal transducer and activator of transcription 3 (STAT3) (93).Another HSP90 client nucleophosmin/anaplastic lymphoma kinase (NPM/ALK) showed to induce PD-L1 via STAT3 activation in T cell lymphoma cells (94).It has been also shown that the spliced isoform of HSP90 co-chaperone FKBP51 regulates the expression of glycosylated PD-L1 in glioma cells (95).Combination of HSP90 inhibitor ganetespib and anti-CTLA-4 associated with an increase in the frequency of CD8 + T cells in mice and decrease in T regs (96).Mechanistically, HSP90 inhibitor upregulates interferon response genes, leading to T cell-mediated killing of melanoma cells (96).
Using mass spectrometry-based proteome profiling several studies showed that various types of immune cells, including NK, T, dendritic cells, platelets, and neutrophils can secrete HSP90s and their cognate co-chaperones in EVs (summarized in Figure 4) (62).Overexpression of HSP90 in hypoxic macrophage-derived exosomes inhibited Hippo signaling pathway, leading to colorectal cancer progression (102).Heat shock and anti-cancer drugs significantly upregulate exosomes release (103).Exosomes secreted by mouse B cell lymphoma cells after heat shock showed elevated expression of HSP90, HSP60 and MHC I, MHC II, CD40, CD86, RANTES and IL-1b (104, 105).These exosomes stimulate DC maturation and more potently induce CTL responses (104).It has also been shown that HSP-bearing exosomes secreted by human hepatocellular carcinoma cells stimulate NK cell cytotoxicity and granzyme B secretion (103).Triple deletion of CDC37, HSP90a and HSP90b diminished EV-driven malignancy progression and macrophage M2 polarization (106).
HSP90 is also involved in the modulation of another form of regulated cell death necroptosis (108,113).Jacobsen and coworkers demonstrated that HSP90 inhibitors block necroptosis by downregulating MLKL expression and membrane translocation (113).Several studies reported that HSP90 inhibitors impact RIP1 stability and function (114-117).A complex consisting of HSP90 and C DC 37 is re quired f or RIP 3 activation during necroptosis (118).

HSP90 in sustained proliferation
Recent studies have reported that HSP90 regulates the activity of tumor suppressor p53 by interacting with its DNA binding domain (128).HSP90 stabilizes mutant p53 in cancer cells leading to uncontrolled proliferation of tumor cells (129, 130).HSP90 also stabilizes the epidermal growth factor receptor (EGFR) tumor cells (129).HSP90 inhibition decreases total and phosphorylated EGFR and suppresses the proliferation of resistant cancer cells (131).In addition, HSP90 activity is essential for ErbB2/HER, v-Src, c-Src, BCR-ABL, Raf1, and other kinases which are known to promote proliferation and survival of cancer cells (132).

HSP90 in replicative immortality
Holt and colleagues demonstrated that HSP90 and its cochaperone p23 associate with human telomerase reverse transcriptase and are required for efficient assembly of functional telomerase (136).HSP90 inhibitor geldanamycin inhibited the assembly of active telomerase in vitro and in vivo (136).Further biochemical studies demonstrated that HSP90 is critical for hTERT folding and stabilization of the assembled telomerase complex (137).HSP90 is also important for the maintenance of telomere length as overexpression of HSP90 associates with telomere shortening (138).In addition, HSP90 promotes telomerase DNA binding (139).Telomere dysfunction may also induce senescence (140).Indeed, Zhong and colleagues demonstrated that an increase in extracellular HSP90a promotes fibroblast senescence by activating TGFb (141).HSP90 inhibitors downregulate phosphorylated form of AKT, leading to apoptosis of senescent cells (142).These data suggest that HSP90 favors tumor growth by modulating telomerase and senescence.

HSP90 in angiogenesis
Song and colleagues reported that HSP90a promotes angiogenesis via stabilizing activated matrix metalloproteinase 2 (MMP-2) (143).Further studies showed that HSP90 also stabilizes macrophage migration inhibitory factor (MIF), which acts as an angiogenesis promoting factor during neoplastic transformation (144,145).Dong et al. demonstrated that breast cancer cells secrete HSP90a to survive under hypoxia (146).HSP90 inhibitor AT-533 has been reported to inhibit growth and angiogenesis by suppressing the HIF-1a/VEGF pathway in hypoxic breast cancer cells (147).These cells also secrete a splice variant VEGF 90K which binds HSP90 on the surface of microvesicles further promoting angiogenesis (148).HSP90/phosphorylated IKK-rich extracellular vesicles from hypoxic melanoma activate pro-angiogenic melanoma-associated fibroblasts (MAFs) via the NF-kB/CXCL1 axis (149).Furthermore, C-terminal HSP90 inhibitor SL-145 has been shown to inhibit growth and angiogenesis by dysregulating JAK2/STAT3 signaling pathway in triple negative breast cancer cells (150).

HSP90 in invasion and metastasis
Extracellular HSP90 interacts with LRP1 (also known as CD91) to induce ERK and MMP-2/9 activation, leading to E-cadherin inhibition and the initiation of EMT in prostate cancer cells (51,151).Furthermore, extracellular HSP90 secreted by these cells upregulates the expression of stem-like markers, promoting self-renewal (152).HSP90 interaction with LRP1 leads to the increased expression of phosphorylated IKKa/b and NF-kB resulting in the induction of TCF12, which in turn decreases Ecadherin and promotes colorectal cancer EMT, migration and invasion (153).HSP90b also associates with LRP5, promoting EMT via Akt and Wnt/b-catenin signaling (12, 154).In metastatic breast cancer cells, HIF-1a downregulation inhibits HSP90a secretion and invasion (155).GRP94, an ER paralog of HSP90 may also promote invasion and metastasis via the regulation of its client GARP, which is critical for the membrane expression of TGFb (156).

HSP90 therapies targeting cancer 6.1 HSP90 inhibitors in cancer clinical trials
Owing to the importance of HSP90 in cancer, it has become an attractive target for anti-cancer therapies.HSP90 inhibitors in clinical trials are summarized in Table 1.Several clinical trials assessed HSP90 inhibitor-linked to verteporfin (HS-201, for imaging and detection of solid tumors.Currently, there are no FDA-approved HSP90 inhibitors.The low effectiveness of HSP90 inhibitors in clinical trials may be attributed to drug-related toxicity and limited efficacy.Insufficient isoform selectivity has been considered as one of the main reasons for these failures.

HSP90 vaccines
The ability of HSP90-peptide complexes to activate both CD8 + and CD4 + T cells led to the development of HSP90-based vaccines (3,157).Innovative approach was proposed by Yamazaki and colleagues who generated a secretory form of ER HSP90 where HSP90 ER (gp96) KDEL retention signal was deleted and replaced with the Fc portion of IgG1, thus imitating necrotic cell death release of HSPs (158).Immunization of mice with tumor cells secreting gp96-Ig resulted in tumor rejection in vivo which was primarily dependent on CD8 + T cells (158,159).Gp96-Ig vaccine, also called Viagenpumatucel-L or HS-110 was further assessed in phase I (NCT00503568) and phase II (NCT02117024) clinical trials in patients with non-small cell lung carcinoma.Gp96-Ig was also assessed in combination with anti-PD-1inhibitor Nivolumab (NCT02439450) and has shown to be well-tolerated and improve overall survival of PD-L1 + patients with advanced lung cancer (160, 161).Crane and colleagues prepared autologous gp96-peptide complexes to immunize patients with recurrent glioblastoma in phase I trial (162).Re-stimulation of peripheral blood leukocytes with autologous gp96 led to increase in IFNg (162).Autologous gp96 prepared from resected tumors in combination with standard radiation and chemotherapy improved overall survival in glioblastoma patients with low expression of PD-L1 + on peripheral myeloid CD45 + CD11b + cells (163).Interestingly, dendritic cells (DCs) pulsed with tumor-derived gp96 showed anti-tumor effect which was significantly dependent on NK and CD8 T cells (164).Multi-chaperone vaccine called "chaperone-rich cell lysate" (CRCL) contains several chaperones, including HSP70, HSP90, gp96 and calreticulin showed to activate DCs and upregulate the expression of CD40, MHC II, IL-12, CD70, iNOS and NF-kB and enhance the phosphorylation of STAT1,STAT5, ERK1/2 and AKT (165,166).CRCL-stimulated DCs and macrophages resisted the suppressive activity of T regulatory cells (167).Notably, depletion of chaperones from CRCL led to the decrease in IFNg production by splenocytes (165).Similar to T cells, CRCL has also been shown to stimulate IFNg, TNFa, RANTES production and the activation of STAT1 and NF-kB by NK cells (168).
Immunization of mice with another multi-chaperone vaccine purified from the mouse sarcoma cell line S180 containing the mixture of HSP60, HSP70, HSP110 and gp96 (mHSP/peptide vaccine) in combination with cyclophosphamide and IL-12 suppressed tumor growth and improved long-term survival (169).Further studies have shown that mHSP/peptide vaccine containing HSP70, HSP90 and gp96 showed superior anti-tumor effect than gp96/peptide vaccine (170).PD-L1 inhibitor in combination with tumor-derived mHSP/peptide vaccine induced the section of IFNg, TNFa, IL-10 and IL-2 on day 14 th whereas on day 28 th combinational treatment led to decrease production of IFNg, IL-2 and IL-10 (170).

Conclusion
HSP90 molecular chaperones are abundantly expressed in cancer, leading to tumor growth and survival via the modulation of various hallmarks of cancer, including sustained proliferation, deregulation of cellular energetics, unlimited replicative potential, tumor immunity, angiogenesis, metastasis and invasion.Given HSP90 ability to promote growth and survival of tumor cells by regulating a wide range of processes and enabling hallmarks of cancer, various HSP90 inhibitors entered clinical trials.Based on the ability of HSP90 to elicit anti-tumor response, several HSP90-based immunotherapies were developed.Further elucidating the complex role of HSP90 in cancer may provide new opportunities for the diagnosis and treatment of cancer patients.

TABLE 1
HSP90 inhibitors in cancer clinical trials.